Bacterial fitness in chronic wounds appears to be mediated by the capacity for high-density growth, not virulence or biofilm functions.

While much is known about acute infection pathogenesis, the understanding of chronic infections has lagged. Here we sought to identify the genes and functions that mediate fitness of the pathogen Pseudomonas aeruginosa in chronic wound infections, and to better understand the selective environment in wounds. We found that clinical isolates from chronic human wounds were frequently defective in virulence functions and biofilm formation, and that many virulence and biofilm formation genes were not required for bacterial fitness in experimental mouse wounds. In contrast, genes involved in anaerobic growth, some metabolic and energy pathways, and membrane integrity were critical. Consistent with these findings, the fitness characteristics of some wound impaired-mutants could be represented by anaerobic, oxidative, and membrane-stress conditions ex vivo, and more comprehensively by high-density bacterial growth conditions, in the absence of a host. These data shed light on the bacterial functions needed in chronic wound infections, the nature of stresses applied to bacteria at chronic infection sites, and suggest therapeutic targets that might compromise wound infection pathogenesis.

The association of psoriasiform rash with anti-tumor necrosis factor (anti-TNF) therapy in inflammatory bowel disease: a single academic center case series.

BACKGROUND & AIMS: Anti-tumor necrosis factors (anti-TNF) including infliximab, adalimumab and certolizumab pegol are used to treat Crohn's disease (CD) and ulcerative colitis (UC). Paradoxically, while also indicated for the treatment of psoriasis, anti-TNF therapy has been associated with development of psoriasiform lesions in IBD patients and can compel discontinuation of therapy. We aim to investigate IBD patient, clinical characteristics, and frequency for the development of and outcomes associated with anti-TNF induced psoriasiform rash. METHODS: We identify IBD patients on anti-TNFs with an onset of a psoriasiform rash. Patient characteristics, duration of anti-TNF, concomitant immunosuppressants, lesion distribution, and outcomes of rash are described. RESULTS: Of 1004 IBD patients with exposure to anti-TNF therapy, 27 patients (2.7%) developed psoriasiform lesions. Psoriasiform rash cases stratified by biologic use were 1.3% for infliximab, 4.1% for adalimumab, and 6.4% for certolizumab. Average time on treatment (206.3weeks) and time on treatment until onset of psoriasiform lesions (126.9weeks) was significantly higher in the infliximab group. The adalimumab group had the highest need for treatment discontinuation (60%). The majority (59.3%) of patients were able to maintain on anti-TNFs despite rash onset. Among patients that required discontinuation (40.7%), the majority experienced improvement with a subsequent anti-TNF (66.7%). CONCLUSION: 27 cases of anti-TNF associated psoriasiform lesions are reported. Discontinuation of anti-TNF treatment is unnecessary in the majority. Dermatologic improvement was achieved in the majority with a subsequent anti-TNF, suggesting anti-TNF induced psoriasiform rash is not necessarily a class effect.

Cutaneous angiosarcoma clinically presenting as progressive solid facial edema in a 43-year-old male.

Cutaneous angiosarcoma of the head and neck is a rare, highly malignant neoplasm; prognosis is heavily influenced by tumor size, resectability, and stage at initial diagnosis. Most patients present with one to several erythematous to violaceous patches, plaques, or nodules. However, the clinical presentation is highly variable and leads to delayed diagnosis. We report cutaneous angiosarcoma in a 43-year-old man who presented with an 11-month history of progressive solid (non-pitting) edema involving his entire face, scalp, eyelids, and neck without characteristic clinical features of cutaneous angiosarcoma. A skin biopsy had shown non-specific findings consistent with solid facial edema or rosacea. Various etiologies were considered but there was no significant improvement after directed medical therapy. Repeat skin biopsies revealed angiosarcoma involving the dermis and sub-cutis. Computed tomography (CT) of the chest showed multiple lung nodules bilaterally and a lytic lesion in the T6 vertebra consistent with metastases. He was treated with single agent chemotherapy (paclitaxel), and had a partial response that restored his ability to open both eyes spontaneously; However, his edema has recently progressed 7 months after diagnosis. This is a rare example of cutaneous angiosarcoma presenting as progressive solid facial edema, which underscores the diverse range of clinical manifestations associated with this neoplasm.

Biofilms and Inflammation in Chronic Wounds.

SIGNIFICANCE: The incidence, cost, morbidity, and mortality associated with non-healing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair. RECENT ADVANCES: Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity. CRITICAL ISSUES: When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host. FUTURE DIRECTIONS: Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy.

Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

Time course study of delayed wound healing in a biofilm-challenged diabetic mouse model.

Bacterial biofilm has been shown to play a role in delaying wound healing of chronic wounds, a major medical problem that results in significant health care burden. A reproducible animal model could be very valuable for studying the mechanism and management of chronic wounds. Our previous work showed that Pseudomonas aeruginosa (PAO1) biofilm challenge on wounds in diabetic (db/db) mice significantly delayed wound healing. In this wound time course study, we further characterize the bacterial burden, delayed wound healing, and certain aspects of the host inflammatory response in the PAO1 biofilm-challenged db/db mouse model. PAO1 biofilms were transferred onto 2-day-old wounds created on the dorsal surface of db/db mice. Control wounds without biofilm challenge healed by 4 weeks, consistent with previous studies; none of the biofilm-challenged wounds healed by 4 weeks. Of the biofilm-challenged wounds, 64% healed by 6 weeks, and all of the biofilm-challenged wounds healed by 8 weeks. During the wound-healing process, P. aeruginosa was gradually cleared from the wounds while the presence of Staphylococcus aureus (part of the normal mouse skin flora) increased. Scabs from all unhealed wounds contained 10(7) P. aeruginosa, which was 100-fold higher than the counts isolated from wound beds (i.e., 99% of the P. aeruginosa was in the scab). Histology and genetic analysis showed proliferative epidermis, deficient vascularization, and increased inflammatory cytokines. Hypoxia inducible factor expression increased threefold in 4-week wounds. In summary, our study shows that biofilm-challenged wounds typically heal in approximately 6 weeks, at least 2 weeks longer than nonbiofilm-challenged normal wounds. These data suggest that this delayed wound healing model enables the in vivo study of bacterial biofilm responses to host defenses and the effects of biofilms on host wound healing pathways. It may also be used to test antibiofilm strategies for treating chronic wounds.

Differential effects of planktonic and biofilm MRSA on human fibroblasts.

Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-ß1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

Quantifying the effect of pore size and surface treatment on epidermal incorporation into percutaneously implanted sphere-templated porous biomaterials in mice.

The sinus between skin and a percutaneous medical device is often a portal for infection. Epidermal integration into an optimized porous biomaterial could seal this sinus. In this study, we measured epithelial ingrowth into rods of sphere-templated porous poly(2-hydroxyethyl methacrylate) implanted percutaneously in mice. The rods contained spherical 20-, 40-, or 60-µm pores with and without surface modification. Epithelial migration was measured 3, 7, and 14 days post-implantation utilizing immunohistochemistry for pankeratins and image analysis. Our global results showed average keratinocyte migration distances of 81 ± 16.85 µm (SD). Migration was shorter through 20-µm pores (69.32 ± 21.73) compared with 40 and 60 µm (87.04 ± 13.38 µm and 86.63 ± 8.31 µm, respectively). Migration was unaffected by 1,1' carbonyldiimidazole surface modification without considering factors of pore size and healing duration. Epithelial integration occurred quickly showing an average migration distance of 74.13 ± 12.54 µm after 3 days without significant progression over time. These data show that the epidermis closes the sinus within 3 days, migrates into the biomaterial (an average of 11% of total rod diameter), and stops. This process forms an integrated epithelial collar without evidence of marsupialization or permigration.

Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes.

BACKGROUND: Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. RESULTS: The impact of S. aureus soluble products in biofilm-conditioned medium (BCM) or in planktonic-conditioned medium (PCM) on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. CONCLUSIONS: Collectively the results indicate that S. aureus biofilms induce a distinct inflammatory response compared to their planktonic counterparts. The differential gene expression and production of inflammatory cytokines by biofilm and planktonic cultures in keratinocytes could have implications for the formation and persistence of chronic wounds. The formation of a biofilm should be considered in any study investigating host response to bacteria.

Resident bacterial flora in the skin of C57BL/6 mice housed under SPF conditions.

Research in cutaneous biology frequently involves models that use mice housed in SPF conditions. Little information is available concerning the species of bacteria that normally inhabit the skin of these mice. The aim of this study was to characterize the bacterial skin flora of mice housed under SPF conditions. Skin biopsies from C57BL/6 mice under normal and surgically prepped conditions were both cultured and analyzed by using DNA extraction and sequencing. The species isolated most commonly from culture were staphylococci. Coagulase-negative staphylococci were isolated more frequently than was Staphylococcus aureus. Molecular sequencing yielded several additional organisms not found by culture. Overall, culturing of isolates yielded 14 species of bacteria, and molecular sequencing identified another 6 species. Investigators conducting cutaneous research in mouse models should aware of the cutaneous bacterial flora present on these mice.

Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds.

Chronic wounds are a major clinical problem that lead to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models. The goal of this study was to create a reproducible chronic wound model in diabetic mice by the application of bacterial biofilm. Six-millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms 2 days postwounding, and covered with semiocclusive dressings for 2 weeks. Most of the control wounds were epithelialized by 28 days postwounding. In contrast, none of biofilm-challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged wounds-all indicators of an inflammatory nonhealing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality, and demonstrated delayed wound healing following a biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing.

Diagnosis of mycosis fungoides with different algorithmic approaches.

BACKGROUND: Algorithmic scoring approaches for evaluating early cases of mycosis fungoides (MF) provide a degree of diagnostic standardization. At the UWMC, biopsies from clinically concerning cases for MF are individually reviewed by a panel of pathologists and an average score is assigned to each biopsy reflecting the degree of suspicion for a diagnosis of MF; however, such an approach may not be practical outside of an academic center. METHODS: 78 cases characterized in our institution, with the diagnosis confirmed by clinicopathologic correlation/clinical follow-up were evaluated with two different algorithms, based entirely on histologic evaluation (Guitart algorithm) and partial implementation of the ISCL algorithm evaluating histology, immunohistochemistry and T-cell clonality. RESULTS: A receiver operating characteristic curve comparing the results of the two approaches in early MF cases showed no statistical difference between the areas under the two curves. Increased stages of MF showed variable loss of T-cell antigens by immunohistochemistry and higher rates of detectable clonality. CONCLUSION: We could not document a statistically significant advantage of adding ancillary immunohistochemical and molecular testing to careful histologic evaluation in the workup of suspected cases of early MF. A systemic approach to histologic diagnosis by a single pathologist correlated favorably to the MF panel diagnosis.

Granulomatous dermatitis with pseudoxanthoma elasticum-like changes: report of a case in a patient with cystic fibrosis.

BACKGROUND: There is scant literature that documents pseudoxanthoma elasticum (PXE)-like histologic changes in the setting of inflammatory skin diseases. This article documents granulomatous dermatitis with PXE-like changes in a patient with cystic fibrosis. This is the first report of its kind, to our knowledge. OBSERVATIONS: A 33-year-old woman with cystic fibrosis developed a papular eruption on the flexural surfaces of the upper and lower extremities, which was initially treated with prednisone. A punch biopsy showed granulomatous inflammation and associated PXE-like changes. The combined histologic and clinical findings were most consistent with granuloma annulare. There was no family history of PXE or clinical manifestations of PXE. The rash gradually resolved itself over the next several months. CONCLUSIONS: There are few publications that document PXE-like changes in association with various inflammatory skin conditions. Thus, the clinical significance of this finding remains uncertain. This case and previous reports are discussed in the context of current molecular and genetic knowledge. It is hoped that greater awareness of this phenomenon will promote further investigation and elucidation of the clinical and biologic significance of PXE-like changes observed in biopsies of inflammatory skin disorders.

Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro.

Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies demonstrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insert-supported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendrite-like extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (p< or =0.0004). At 24 hours, biofilm-, BCM-, and PCM-exposed HK all exhibited reduced scratch closure (p< or =0.0001). The results demonstrated that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus.

Ultrastructural localization of integrin subunits beta4 and alpha3 within the migrating epithelial tongue of in vivo human wounds.

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.

Methods to promote Notch signaling at the biomaterial interface and evaluation in a rafted organ culture model.

The Notch signaling pathway is a promising target for controlling cell fate choices at the biomaterial-tissue interface. Building on our previous work in developing Notch-signaling biomaterials, we evaluated various immobilization schemes for Notch ligands, and their effect on human foreskin keratinocytes. A peptide sequence derived from the Jagged-1 DSL-region and immobilized to poly(2-hydroxyethyl methacrylate) (polyHEMA) showed no bioactivity in relation to the Notch-CSL pathway. The full-length Jagged-1 protein immobilized directly to the polyHEMA surface showed activity in signaling the Notch-CSL pathway. However, an indirect affinity immobilization approach yielded a stronger signal. Human keratinocytes plated on bound Jagged-1 showed upregulated involucrin, keratin 10, and loricrin protein expression, with this expression being cell density-dependent. Utilizing a human foreskin rafted organ culture model as a bridge between in vitro and in vivo studies, Jagged-1-modified or control polyHEMA rods were implanted in human foreskin and cultured at the air-medium interface. Keratinocyte proliferation was suppressed and intermediate-stage differentiation promoted in Jagged-1-modified rods compared with control rods. Thus, Notch-signaling biomaterials provide a robust approach to control keratinocyte differentiation and may find application to other progenitor and stem cells.

Models for diabetic wound healing and healing into percutaneous devices.

Delayed healing of chronic ulcers in patients with diabetes and infections resulting from percutaneous medical devices (e.g., vascular access catheters) not only plays a major role in morbidity and mortality, but is a major burden to our healthcare system. In this paper, we review models to study diabetic wound healing and the wound-healing response associated with implanted percutaneous implants.